keeping adherent cells ins suspension culture?

Emir Khatipov khatipovNO at NOuchicago.edu
Thu Apr 11 12:01:47 EST 2002


Wo,
Just a thought, no personal experience. I checked "Animal Cell Culture. A
practical approach" book by Fresney, 1992. Page 78 gives a protocol for
adaptation of the cells to suspension growth. One of the clues I guess is to
use calcium- and magnesium-free media - these ions are required for
attachment. So, the solution for you could be either adapt the cells to
suspended growth, or probably to supplement the media with enough EDTA to
chelate Ca and Mg (each EDTA molecule binds 2 to 4 bivalent cations, if I am
not mistaken). I would probably use EDTA at twice as high molar
concentration as combined concentration of the cations.
Emir


""Wolfgang Schechinger"" <wolfsc at ibms.sinica.edu.tw> wrote in message
news:3CB5D22C.24275.1880419 at localhost...
> Dear all,
>
> How can I overcome this problem:
>
> I have cells expressing an enzyme pinned on the extracellular part of the
> membrane. To assay this enzyme by FACS, I need to trypsinize the cells,
but
> this (just 5 min) reduces the enzyme activity significantly. Detaching the
> cells with EDTA kills too many of them.
>
> What can I do to either
>
> 1) obtain a single cell suspension or
>
> 2) to keep the cells for some time in suspension until some newly
synthesized
> protein is delivered to the membrane?
>
> I welcome your input
>
> Regards,
>
> Wo
> -----
> Dr. Wolfgang Schechinger
> Institute of Biomedical Sciences
> Academia Sinica, Taipei, Taiwan
>
> ---





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